Hyperbolic Substrate Saturation Curves Research Articles

The effect of omega-6 and omega-3 fatty acids on 3H-thymidine incorporation in hepatoma 7288CTC perfused in situ.

Ingestion of diets containing corn oil or marine fish oils is known to increase or decrease, respectively, the growth of transplantable rodent tumours. The active agents in these oils have been identified as linoleic acid (in corn oil) and omega-3 fatty acids (in marine oils), but it is still not known how they influence the tumour growth processes. In these experiments we examined the effects of plasma free omega-6 and omega-3 fatty acids on the rate of 3H-thymidine incorporation in tissue-isolated hepatoma 7288CTC perfused in situ. Host Buffalo rats were fed an essential fatty acid-deficient diet. Plasma and tumours in these animals contained low endogenous levels of both omega-6 and omega-3 fatty acids. Perfusion of these tumours for 2 h with donor whole blood containing added omega-6 free fatty acids, including 0.5 mM linoleic (C18:2,N-6), gamma-linolenic (C18:3,N-6), dihomo-gamma-linolenic (C20:3,N-6) or arachidonic acids (C20:4,N-6), increased the rate of 3H-thymidine incorporation. Linoleic acid was about three times more effective than the other omega-6 fatty acids. Typical hyperbolic substrate-saturation curves were observed as the plasma free linoleate or arachidonate concentration was increased. When perfused alone plasma free omega-3 fatty acids had no effect on tumour 3H-thymidine incorporation, but in the presence of linoleic acid the omega-3 fatty acids, alpha-linolenic (C18:3,N-3) and eicosapentaenoic (C20:5,N-3), competitively inhibited both tumour linoleate uptake and the stimulative effect on 3H-thymidine incorporation. The results suggest that the ambient plasma free linoleic and arachidonic acid concentrations in host arterial blood directly influence the rate of tumour DNA synthesis. Plasma free omega-3 fatty acids appear to modulate the effect of linoleic acid by competitively inhibiting its uptake. These relationships could explain the actions of dietary linoleic and omega-3 fatty acids on tumour growth in vivo.

PARTIAL PURIFICATION OF SOLUBLE 5′-NUCLEOTIDASE FROM RABBIT HEART: 39

Cytosolic 5′-nucleotidase from rabbit heart has been purified approximately 40-fold to a specific activity of 0.6 μmol/mg protein/min. At this stage of purification, the enzyme is free of other phosphatases and is stable to repeated cycles of freezing and thawing over a 3 months period. Soluble 5′-nucleotidase has an absolute requirement for divalent metal ions and is activated by ATP, GTP, and ADP. Activation by these nucleotides causes a shift from a sigmoidal to a hyperbolic substrate saturation curve. ATP (1 mM) increases the Kapp of the enzyme for AMP about 5-fold. Activation by GTP is similar. Activation by ADP also increases Kapp, but high concentrations of ADP cause a decrease in Vmax. AMP is the preferred substrate in terms of Vmax/Kapp, with AMP>UMP>GMP>IMP. Nucleoside monophosphates other than AMP are inhibitory at high substrate concentrations. The fully activated enzyme exhibits a Kapp for AMP of 2.5 mM. The enzyme purified by us has properties different from the soluble liver enzyme described by Van Den Berghe et al. (Biochem. J. (1977) 162, 611) and from the plasma membrane enzyme from rat heart described by Naito & Lowenstein (Biochem. (1981) 20, 5188) with respect to both substrate specificity and regulatory properties.

Differences in the sensitivity of carnitine palmitoyltransferase to inhibition by malonyl-CoA are due to differences in Ki values.

The hepatic carnitine palmitoyltransferase that is present on the outer surface of the mitochondrial inner membrane demonstrates hyperbolic substrate saturation curves with oleoyl-CoA in both fasted and fed rats. However, the addition of malonyl-CoA resulted in sigmoid substrate saturation curves, suggesting that malonyl-CoA induced the cooperative behavior. There was more of the outer carnitine palmitoyltransferase in liver mitochondria derived from fasted rats and that enzyme had a much greater Ki for malonyl-CoA than the enzyme from fed rats, but the Km values were apparently not different. The Dixon plot with mitochondria from fed rats, but not fasted rats, was curved upward, indicating cooperative inhibition by malonyl-CoA. Carnitine palmitoyltransferase of heart mitochondria had a Ki for malonyl-CoA that was much less than that of the liver enzyme and it did not change on fasting. Furthermore, no evidence for cooperative inhibition was found in the heart. The results of these studies indicate that carnitine palmitoyltransferase is not subject to substrate cooperativity and that malonyl-CoA is not a simple competitive inhibitor of this enzyme but inhibits by a mechanism involving cooperative inhibition. The fasting-feeding cycle induces changes in the liver enzyme that alter its affinity for malonyl-CoA without changing its affinity for its acyl-CoA substrate. Carnitine palmitoyltransferase from heart appears to be different from that of liver and is apparently not subject to the same control mechanisms.

Separation and characterization of two chorismate-mutase isoenzymes from Nicotiana silvestris

Two isoenzymes of chorismate mutase (EC 5.4.99.5) were isolated and partially purified from leaves of diploid (2n=24) Nicotiana silvestris Speg. et Comes and from isogenic cells in a suspension culture originally established from haploid tissue. An isoenzyme denoted CM-1 (M r=52,000) accounted for the major fraction of total activity recovered from suspension-cultured cells, while isoenzyme CM-2 (M r=65,000) represented the major fraction of activity recovered from green leaf tissue. The ratio of isoenzyme levels from these two sources differed more than 20-fold. The subcellular location of isoenzyme CM-1 is known to be in the chloroplasts of green leaves or in proplastids of cultured cells, while isoenzyme CM-2 is located in the cytosol. Both isoenzymes were stable during partial purification, possessed broad pH optima for catalysis between 6.0 and 8.0, and were active without denaturation at temperatures at least as high as 45° C. Thiol reagents were unnecessary for either stability or activity of both isoenzymes. The affinity of isoenzyme CM-2 for substrate (K m=0.24 mM) was almost an order of magnitude better than that of CM-1. The kinetic behavior of isoenzyme CM-1 was influenced by pH, while that of isoenzyme CM-2 was not. At pH 7.2, hyperbolic substrate-saturation curves (K m=1.7 mM) were obtained for isoenzyme CM-1. At pH 6.1, however, isoenzyme CM-1 displayed relatively weak positive cooperativity, Hill plots yielding an n value of 1.2 At pH 6.1 the half-saturation ([S]0.5) value was 2.5 mM.

An experimental test for cyclic versus linear transport models. The mechanisms of glucose and choline transport in erythrocytes.

Numerous mechanisms have been suggested to explain transport across biological membranes, all of which fall into one or the other of two distinct categories. In some, substrate sites in the free carrier are simultaneously exposed on the two membrane surfaces, while in others a substrate site is alternately exposed on opposite sides. Either group could account for active and facilitated transport, as well as for accelerated exchange, countertransport, and hyperbolic substrate saturation curves. A simple kinetic test is described here which distinguishes between these two classes. The test depends on measurements of transport rates in the presence of reversible competitive inhibitors inside and outside the cell. Experiments are reported on the glucose system of erythrocytes involving the inhibitors phloretin and cytochalasin B, and on the choline system of the same cells, with the nontransported substrate analogs dimethyl-n-pentyl(2-hydroxyethyl)ammonium ion and 2-di-butylaminoethanol. The results are in agreement with the single site-exposure models, which include the classical carrier, and incompatible with the dual site-exposure models. The mechanisms in the latter group are therefore rejected as explanations for glucose or choline transport.

Hybrid aspartate transcarbamoylase containing cross-linked subunits

The role of conformational changes and subunit interactions in the allosteric mechanism of aspartate transcarbamoylase was evaluated by studying hybrid enzyme molecules containing cross-linked subunits. Native enzyme was cross-linked with tartryl diazide in the presence and absence of substrate analogues. The two types of modified enzyme derivatives were each dissociated into catalytic (c3) and regulatory (r2) subunits. Hybrids were constructed with modified catalytic subunits and unmodified regulatory subunits of vice versa. Subunits from different derivatives also formed hybrids. All hybrids containing cross-linked catalytic subunits showed hyperbolic substrate saturation curves while cross-linked in the regulatory subunit alone did not abolish cooperativity. The type of cross-linked in the catalytic subunit had a decisive influence on the substrate affinity of the hybrid as well as its response to the allosteric effectors ATP and CTP. However many effects were also dependent on the presence of regulatory subunits. The results implicate a substantial conformational change in the catalytic subunit upon substrate binding and suggest an important role for the c-r interaction in the allosteric mechanism.

Effect of association-dissociation on the catalytic properties of glyceraldehyde 3-phosphate dehydrogenase

The enzymatic activity of d-glyceraldehyde 3-phosphate dehydrogenase depends nonlinearly on protein concentration in the range 3 × 10 −8 to 3 × 10 −6 m. With increasing enzyme concentrations the apparently hyperbolic substrate saturation curves turn into sigmoidal ones. From the kinetic and physicochemical data it is assumed that the enzyme exists as an equilibrium mixture of different oligomeric states. The system is found to be consistent with a model characterized by rapid equilibrium between monomer-dimer-tetramer, the tetramer being inactive, assuming identical intrinsic binding constants for the substrate in the monomer and in the dimer.

Testing transport models with substrates and reversible inhibitors

The kinetic behavior of five models for biological transport, only one of which is based on the classical carrier mechanism, is investigated. All give hyperbolic substrate saturation curves in accord with experimental observations on many systems. Several simple kinetic tests with substrates and competitive inhibitors serve to exclude or confirm proposed models. The tests involve measuring rates of efflux of radioactive substrate in the presence of (i) a competitive inhibitor outside the cell; (ii) inhibitor inside and outside; and (iii) unlabeled substrate outside. Rules for testing hypothetical mechanisms are presented in tables which may be consulted directly, disregarding the mathematical derivation.

Regulation of glucose, fructose and sucrose catabolism in Rhodopseudomonas capsulata.

Regulation of glucose, fructose and sucrose catabolism was studied in Rhodopseudomonas capsulata grown under phototrophic conditions. The sequence of preference for the utilization of the sugar substrates was fructose, glucose, sucrose. The presence of a preferred substrate did not completely suppress the utilization of the less preferred. Glucose-6-phosphate dehydrogenase, the key enzyme of glucose and sucrose catabolism, exhibited sigmoidal substrate saturation curves and was inhibited by phosphoenolpyruvate, whereas 1-phosphofructokinase, the key enzyme of fructose catabolism, exhibited hyperbolic substrate saturation curves and was not inhibited by phosphoenolpyruvate. Since phosphoenolpyruvate is a common intermediate of glucose, fructose and sucrose catabolism, the control of glucose-6-phosphate dehydrogenase may be responsible for the preferential utilization of fructose.

The Regulatory Role of Muscle Pyruvate Kinase in Carbohydrate Metabolism of Invertebrates: A Comparative Study in Catalytic Properties of Enzymes Isolated from Tubifex tubifex (Oligochaeta) and Tenebrio molitor (Coleoptera)

Biochemical properties of the pyruvate kinases (PK) isolated from body-wall tissue of Tubifex tubifex (Oligochaeta) and from larvae of the tenebrionid beetle Tenebrio molitor (Coleoptera) have been investigated. Tubifex PK was characterized by sigmoidal kinetics with phosphoenolpyruvate (PEP) in the presence of Mg²⁺ and by allosteric regulation under the influence of energy charge and fructose-1,6-diphosphate (FDP). No significant effects of alanine were found. In the presence of Mn²⁺, hyperbolic substrate saturation curves were obtained. Tenebrio PK showed hyperbolic kinetics with PEP which were not modified by divalent cations and FDP. Tubifex and Tenebrio PKs display $Q_{10}$ values near 2 at saturating PEP concentrations. The Tubifex enzyme is less thermally stable than the Tenebrio enzyme, a distinction observed between other regulatory and nonregulatory PKs. The regulatory role of Tubifex PK in aerobic/anaerobic transitions is discussed relative to the different regulatory properties found for this PK compared with PKs from other facultatively anaerobic invertebrates.

L-serine dehydratase from Arthrobacter globiformis

1. L-Serine dehydratase (EC 4.2.1.13) was purified 970-fold from glycine-grown Arthrobacter globiformis to a final specific activity of 660micronmol of pyruvate formed/min per mg of protein. 2. The enzyme is specific for L-serine; D-serine, L-threonine and L-cysteine are not attacked. 3. The time-course of pyruvate formation by the purified enzyme, in common with enzyme in crude extracts and throughout the purification, is non-linear. The reaction rate increases progressively for several minutes before becoming constant. The enzyme is activated by preincubation with L-serine and a linear time-course is then obtained. 4. The substrate-saturation curve for L-serine is sigmoid. The value of [S]0.5 varies with protein concentration, from 6.5mM at 23microng/ml to 20mM at 0.23microng/ml. The Hill coefficient remains constant at 2.9.5 The enzyme shows a non-specific requirement for a univalent or bivalent cation. Half-maximal activity is produced by 1.0mM-MgCl2 or by 22.5mM-KCl. 6. L-Cysteine and D-serine act as competitive inhibitors of L-serine dehydratase, with Ki values of 1.2 and 4.9mM respectively. L-Cysteine, at higher concentrations, also causes a slowly developing irreversible inhibition of the enzyme. 7. Inhibition by HgCl2 (5micronM)can be partially reversed in its initial phase by 1mM-L-cysteine, but after 10 min it becomes irreversible. 8. In contrast with the situation in all cell-free preparations, toluene-treated cells of A. globiformis form pyruvate from L-serine at a constant rate from the initiation of the reaction, show a hyperbolic substrate-saturation curve with an apparent Km of 7mM and do not require a cation for activity.

The influence of divalent cations on allosteric behaviour of muscle pyruvate kinase from the sea mussel Mytilus edulis L.

1. 1. Pyruvate kinase of the adductor muscle of the sea mussel displays an absolute requirement for Mg 2+ or Mn 2+. By the use of Ca 2+ or Zn 2+ no enzyme activity is obtained. 2. 2. In the presence of Mn 2+, in contrast to Mg 2+, always hyperbolic substrate saturation curves are obtained. 3. 3. There is evidence that Mn 2+ not only acts by forming an ADP-Mn 2+ complex, but also as an allosteric activator. 4. 4. Ca 2+ is a strong inhibitor but the enzyme becomes less sensitive to this inhibition when Mg 2+ is replaced for Mn 2.

Threonine deaminase from Salmonella typhimurium. Relationship between regulatory sites.

Kinetic analysis of the biosynthetic threonine deaminase, EC 4.2.1.16, from Samonella typhimurium yields hyperbolic substrate saturation curves in the absence of, and higher order substrate saturation curves in the presence of, L-isoleucine. L-Valine reverses this effect of L-isoleucine by restoring the hyperbolic substrate saturation curves. The inhibition of enzyme activity and the reversal of valine stimulation is a function of a second order concentration of L-isoleucine, whereas antagonism of inhibition is a function of first order concentration of valine. The antagonistic effects on enzyme activity of L-isoleucine and of L-valine appear as competitive in diagnostic plots. Threonine deaminase possesses two L-isoleucine binding sites (Kd equals 3.6 muM) and one L-valine binding site (Kd equals 26 muM); the binding of these ligands appear competitive. Exclusion of L-valine requires the binding of 2 molecules of L-isoleucine whereas binding of a single L-valine molecule prevents the binding of 2 L-isoleucine molecules. Cooperative binding of L-isoleucine is not observed under any of the conditions tested. Two cases, expressed in terms of modified Adair equations and based upon the assumption that L-threonine also serves as an activator ligand which binds to the L-valine site, are presented. Case I states that liganding of the activator sites must percede substrate-binding at the active site, and Case II states that the activator site liganding is required solely for reactivation of the L-isoleucine-inhibited enzyme. Analysis of kinetic data by a curve-fitting process suggests that Case II described the relationship between the activator site and the L-isoleucine sites. An enzymatically inactive derivative of threonine deaminase, prepared by reduction with borohydride, binds isoleucine and valine in a manner similar to native holoenzyme. Binding of L-threonine and L-valine to the derivatized enzyme is competitive. The Kd for threonine binding is 3 mM, which is in excellent agreement with the Kd determined by the curve fitting process. It is concluded that the modulation of threonine deaminase activity is wrought by interaction between inhibitor sites and an activator site rather than inhibitor and active sites and that induced transitions rather than concerted transitions more adequately describe the underlying regulatory principle.

Influence of Possible in situ Ionic Environment on Kinetics of Purified Citrate Synthase from an Osmoconformer Sea Anemone, Bunedosoma cavernata

Kinetics of utilization of acetyl coenzyme A by citrate synthase of a sea anemone, an osmoconformer, were compared with those of citrate synthases of various osmoregulators. The Kms of the latter enzymes were substantially increased by higher concentrations of salt and the enzyme exhibited hyperbolic substrate saturation curves. Citrate synthase from sea anemone, on the other hand, exhibited allosteric kinetics and minimal effects of salt on its Km. We suggest that the adaptive advantage of this enzymic property to a sedentary osmoconforming organism such as sea anemone is obvious since the osmoregulating creatures are apparently unable to maintain an appropriate low ionic environment in situ and thus probably the Km of their citrate synthases at a low level.

Formula omitted] fructose-1,6-diphosphate aldolase: Inhibition by sodium pyruvate

Neurospora fructose-1,6-diphosphate aldolase exhibited a hyperbolic substrate saturation curve which changed to sigmoidal in the presence of 0.5 mM sodium pyruvate. The S 0.5 value for fructose-1,6-diphosphate increased from 1.4 mM to 6.6 and 20 mM in the presence of 0.5 and 1.0 mM sodium pyruvate, respectively. The inhibition seems to be cooperative in nature and involves conformational changes. Potassium ions completely blocked the inhibition by sodium pyruvate.

Kinetic properties of cerebral pyruvate kinase.

Partly purified guinea-pig brain pyruvate kinase is not activated by fructose 1,6-diphosphate and gives hyperbolic substrate-saturation curves with phosphoenolpyruvate. It is therefore different from the L-type pyruvate kinase of mammalian liver. Inhibition by MgATP(2-) was competitive for MgADP(-) but not for phosphoenolpyruvate, and the enzyme is therefore different from the M-type pyruvate kinase, which is said to be competitively inhibited by MgATP(2-) with respect to both substrates. The K(i)(MgATP(2-)) value of approx. 8mm for the brain enzyme is higher than the values (about 2mm) reported for the muscle enzyme. Stimulation of enzymic activity was observed at low (1-2mm) concentrations of MgATP(2-). Substrate kinetic constants were K(m) (MgADP(-))=0.47mm, K(m) (phosphoenolpyruvate)=0.08mm. Free Mg(2+) at very high concentrations (over 10mm) was inhibitory (K(i)=20-32mm). Neither ADP(3-) nor 5'-AMP(2-) inhibited the activity. The brain enzyme was concluded to be different from both the M-type and the L-type of other mammalian organs such as muscle and liver.

Kinetic and molecular characteristics of allosteric pyruvate kinase from muscle tissue of the sea mussel Mytilus edulis L

1. 1.At low phosphopyruvate concentrations, fructose 1,6-diphosphate enhances the activity of pyruvate kinase (ATP:pyruvate phosphotransferase, EC 2.7.1.40) from the adductor muscle of sea mussels. At pH 7.6 [phosphopyruvate] - 0.2 mM and [ADP] = 5 mM, half-maximal stimulation is reached with [ Fru-1,6-P 2] = 2 μM; at ph 6.7 it is reached with [ Fru-1,6-P 2] = 15 μM 2. 2.Glucose 1,6-diphosphate enhances enzyme activity but less so than Fru-1,6-P 2 . Cyclic AMP stimulates to a even lesser extent 3. 3.The reaction rate is inhibited when the concentration of the co-substrate ADP is substantially higher than phosphopyruvate. The Lineweaver-Burk plots for ADP at fixed concentrations of phosphopyruvate show no cooperative effects, whilst similar plots for phosphopyruvate at fixed concentrations of ADP all show positive cooperativity 4. 4.Alanine strongly inhibits enzyme activity. At pH 6.7 [phosphopyruvate] = 0.2 mM and [ADP] - 2 mM, 50% inhibition is reached with [alanine] = 0.1 mM; at pH 7.6 it is reached at [alanine] - 1 mM. 0.1 mM Fru-1,6-P 2 counteracts this inhibition giving a hyperbolic substrate-saturation curve 5. 5.ATP inhibition is strongly pH-dependant. At pH 6.7, [phosphopyruvate] = 0.2. mM and [ADP] = 2 mM, 50% inhibition is reached at [ATP] = 0.62 mM. At pH 7.6 there is a little influence of ATP. Fru-1,6-P 2 also cancels this inhibition. Other trinucleotides and AMP inhibit to a less extent 6. 6.The molecular mass of the enzyme was found to be 220 000 ± 10%. On electrofocusing, one main peak is found with pI 6.70. Three smaller peaks have pI values of 6.44, 6.59 and 6.83.

Modulation of guanine deaminase.

1. Guanine deaminases purified from the 15000g supernatant fraction of iso-osmotic sucrose homogenates of rat and mouse liver and brain were tested for the influence of GTP and allantoin. 2. The suffixes A and B were assigned to the isoenzyme fractions eluted from DEAE-cellulose with the lower and the higher molarity of eluent respectively. Isoenzyme A from rat liver, the activity of which showed a sigmoid dependence on substrate saturation, was activated by GTP and inhibited by allantoin. Isoenzyme B, which had a hyperbolic substrate-saturation curve, was not influenced by GTP or allantoin. 3. Isoenzyme A from rat brain, the activity of which had a sigmoid dependence on substrate concentration, was stimulated by GTP. Isoenzyme B, which showed classical Michaelis-Menten kinetics, was inhibited by allantoin. 4. Mouse liver guanine deaminase was not influenced by either GTP or allantoin. 5. Isoenzyme A from mouse brain, which had a hyperbolic substrate-saturation curve, was not influenced by GTP or allantoin but isoenzyme B, with sigmoidal kinetics, was inhibited by allantoin. 6. Mg(2+) activated, or inhibited or did not have an effect on guanine deaminase, depending on the source of the enzyme. 7. The bearing of the above findings on the possible regulation of guanine deaminase activity in vivo is discussed.

Kinetics and regulation of the salt-dependent aspartate transcarbamylase of Halobacterium cutirubrum.

Properties of the aspartate transcarbamylase of the extremly halophilic bacterium Halobacterium cutirubrum, an enzyme that needs high salt concentrations for activity and regulation, were studied in cell-free extracts. The enzyme was stable on prolonged incubation at 4 C in concentrated extracts (50 mg of protein per ml) but not in diluted extracts. Mg(2+) ions and beta-mercaptoethanol stabilized enzyme activity. At salt concentrations below the maximum for activity (3.5 m), the enzyme was rapidly inactivated. Carbamyl phosphate stabilized the enzyme under these conditions; aspartate had a smaller effect. The enzyme was most stable at 0 C; raising or lowering the temperature from this point increased the rate of inactivation. On exposure to lowered salt concentrations, enzyme activity was more sensitive than feedback inhibition. Hyperbolic substrate saturation curves were found for carbamyl phosphate. The K(m) obtained varied with the salt concentration used. With aspartate, sigmoidal curves were found when extracts were assayed immediately after preparation, but hyperbolic curves were obtained with extracts allowed to stand 1 to 2 hr. The presence of cytidine triphosphate (CTP) decreased the V(max) but did not change the K(m); this is thus a V-type enzyme. Low concentrations of succinate activated the enzyme, in the presence and absence of CTP; higher concentrations did not affect its activity. CTP increased the activation energy of the enzyme in 3.5 m salt but decreased it in 2.0 m salt. At both salt concentrations, the sensitivity of the enzyme to feedback inhibition diminished with increasing temperatures. Gel chromatography suggested that the enzyme in crude extracts had a molecular weight of 160,000. Precipitating the enzyme with polyethylene glycol decreased the molecular weight to 34,000, and this activity was no longer sensitive to CTP. The presence of either substrate of the enzyme during polyethylene glycol treatment prevented dissociation of the enzyme and loss of feedback inhibition. Thus, as with other aspartate transcarbamylases, association of subunits seems to be required for regulation of activity by end product.

Aspartate transcarbamoylase from Phaseolus aureus. Partial purification and properties

1. Aspartate transcarbamoylase from 4-day-old radicles of Phaseolus aureus was purified 190-fold by (NH(4))(2)SO(4) fractionation, DEAE-cellulose and DEAE-Sephadex chromatography and Sephadex-gel filtration. The partially purified enzyme, which required P(i) for maximum stability, had an apparent molecular weight of 83000+/-5000. 2. Uridine nucleotides were found to inhibit the activity; UMP was the most potent inhibitor, followed by UDP and UTP. No other nucleotide was found to affect the enzyme, nor could UMP inhibition be overcome by adding another nucleotide. Aspartate gives a hyperbolic substrate-saturation curve, both with and without UMP. The nucleotide inhibitor is non-competitive with respect to this substrate. Carbamoyl phosphate also yields a hyperbolic substrate-saturation curve in the absence of feedback inhibitor, but when UMP is added a sigmoidal pattern results, and the inhibition is competitive with carbamoyl phosphate. 3. The degree of inhibition by UMP is not affected by p-chloromercuribenzoate, urea, mild heat pretreatment or change in pH over the range 8.5-10.5, but is affected by temperature. 4. The aspartate analogue, succinate, both activates and inhibits the reaction, depending on the concentrations of aspartate and succinate used. 5. Kinetic studies with the partially purified enzyme showed that the K(m) for carbamoyl phosphate (0.091 mm) is much lower than that for aspartate (1.7mm). A sequential reaction mechanism was inferred from product-inhibition kinetics, with carbamoyl phosphate binding to the enzyme before aspartate, and the product, carbamoylaspartate, being released ahead of P(i). Initial-velocity studies gave a set of parallel reciprocal plots, compatible with an essentially irreversible step occurring before the binding of aspartate.